Periodic Reporting for period 1 - PRONTO (self-PoweRed cONductimeter for digiTalization of rapid mOlecular diagnostics)
Reporting period: 2023-06-01 to 2024-11-30
Infectious diseases still impose a great health burden. Major diseases such as HIV/AIDS, malaria, hepatitis or tuberculosis cause millions of annual deaths and an estimate disease treatment cost of US$8 trillion. STIs can have serious consequences beyond the immediate impact of the infection itself. In this scenario, quality diagnostics remains one of the key factors that allows for quick patient treatment and prevention of disease further spread. While PCR-based tests proved to be a reliable and high-sensitive gold-standard performed at sophisticated laboratories with skilled personnel at a high cost per test, antigenic tests offered a rapid detection of virus at the point-of-need but, detected the virus presence only after several days of disease incubation. During the SARS-Covid pandemic, it became clear that there is a technological need to develop rapid tests that combines not only PCR sensitivity but also the usability and price of lateral flow antigenic alternatives for pathogens with pandemic potential. In this sense, the development of isothermal nucleic acid amplification (NAA) techniques opened a promising road towards portable solutions. However, until today, the integration of new functionalities entailing NAA and digital readout approaches into Point-Of-Care (POC) solutions generated too costly and complex devices to become an universal tool.
PRONTO proposed a novel low cost, sustainable and self-powered readout system for NAA detection based on conductimetry. The key component consisted on a paper battery that is activated upon the addition of the fluid to be measured. This allows merging sensor and battery in a single element – a battery sensor - whose generated power is directly related to the conductivity of the sample to be analysed. The implementation of the self-powered conductivity sensor readout in nucleic acid testing can enable a radical simplification of electronic components required to sense and digitalize the signal. Therefore, PRONTO approach has a clear potential to deploy a simple and low-cost test while offering unequivocal result, as it delivers an output voltage signal that can be easily converted into a digital output. The effective realization of such approach has the potential to overcome the current cost and environmental barriers that state-of-the art developments impose and become a global solution for diagnostics.
PRONTO proposed a novel low cost, sustainable and self-powered readout system for NAA detection based on conductimetry. The key component consisted on a paper battery that is activated upon the addition of the fluid to be measured. This allows merging sensor and battery in a single element – a battery sensor - whose generated power is directly related to the conductivity of the sample to be analysed. The implementation of the self-powered conductivity sensor readout in nucleic acid testing can enable a radical simplification of electronic components required to sense and digitalize the signal. Therefore, PRONTO approach has a clear potential to deploy a simple and low-cost test while offering unequivocal result, as it delivers an output voltage signal that can be easily converted into a digital output. The effective realization of such approach has the potential to overcome the current cost and environmental barriers that state-of-the art developments impose and become a global solution for diagnostics.
PRONTO project consisted of exploring the viability of detecting changes in ionic conductivity taking place during the amplification of a specific nucleic acid sequence of SARS-Covid and using a battery sensor to produce the readout signal. According to the literature, the consumption of primers and dNTPs, plus the formation of magnesium precipitates due to the interaction of magnesium ions and generated pyrophosphates, led to a detectable decrease of the overall ionic strength of the solution during amplification.
The project departed from an already stablished knowledge on self-powered conductimetric sensors and explored a chemistry, based on a Zn anode and Ag/AgCl cathode screen-printed electrodes. The battery was designed to operate with very small liquid volumes, compatible with the 25 µl required for NAA of SARS-Covid with LAMP chemistry.
EIS technique was used to determine the changes in ionic conductivity during the LAMP amplification process with a set of microfabricated electrodes. Results showed that conductivity values of LAMP chemistry were significantly higher than those reported in the literature and that there were no detectable changes in conductivity that could be attributed to nucleic acid amplification, even at extremely high viral loads. Therefore, this approach was considered not viable. Nevertheless, the battery developed in this project - which was fully printed – showed excellent performance both as power source and conductimetric sensor, which remains open to provide a low-cost and flexible self-powered device for other further applications.
In view of the technical negative results in conductimetry, we decided to explore another new alternative: the viability of developing a pH-sensitive battery, which consists of implementing a battery with a redox chemistry that involves either protons or hydroxide ions in its reactions. With this approach, the output power of the battery depended the pH of the solution used for its activation. This was used to detect the pH changes taking place upon nucleic acid amplification in the non-buffered LAMP amplification version of the SARS-Covid commercial kit. This approach, which required the substitution of the AgCl cathode by a pH-sensitive Pt thin layer, showed that it is possible to detect such pH changes with the novel device, although the detection process itself at neutral pH diminishes the proton concentration at the electrode surroundings.
As a final proof-of-concept, the battery was tested with negative and positive non-buffered colorimetric LAMP kits and the results were promising: the tested samples yielded a clear differentiation in response before and after the amplification protocol. Therefore, this project opens a new avenue for the development of pH-sensitive self-powered devices, with a wide range of redox reactions to be explored and application where pH changes indicate the occurrence of a relevant biological event.
The project departed from an already stablished knowledge on self-powered conductimetric sensors and explored a chemistry, based on a Zn anode and Ag/AgCl cathode screen-printed electrodes. The battery was designed to operate with very small liquid volumes, compatible with the 25 µl required for NAA of SARS-Covid with LAMP chemistry.
EIS technique was used to determine the changes in ionic conductivity during the LAMP amplification process with a set of microfabricated electrodes. Results showed that conductivity values of LAMP chemistry were significantly higher than those reported in the literature and that there were no detectable changes in conductivity that could be attributed to nucleic acid amplification, even at extremely high viral loads. Therefore, this approach was considered not viable. Nevertheless, the battery developed in this project - which was fully printed – showed excellent performance both as power source and conductimetric sensor, which remains open to provide a low-cost and flexible self-powered device for other further applications.
In view of the technical negative results in conductimetry, we decided to explore another new alternative: the viability of developing a pH-sensitive battery, which consists of implementing a battery with a redox chemistry that involves either protons or hydroxide ions in its reactions. With this approach, the output power of the battery depended the pH of the solution used for its activation. This was used to detect the pH changes taking place upon nucleic acid amplification in the non-buffered LAMP amplification version of the SARS-Covid commercial kit. This approach, which required the substitution of the AgCl cathode by a pH-sensitive Pt thin layer, showed that it is possible to detect such pH changes with the novel device, although the detection process itself at neutral pH diminishes the proton concentration at the electrode surroundings.
As a final proof-of-concept, the battery was tested with negative and positive non-buffered colorimetric LAMP kits and the results were promising: the tested samples yielded a clear differentiation in response before and after the amplification protocol. Therefore, this project opens a new avenue for the development of pH-sensitive self-powered devices, with a wide range of redox reactions to be explored and application where pH changes indicate the occurrence of a relevant biological event.
1) Despite the initial proposed path for self-powering detection based on conductimetry was not successful, this project opens a new avenue for the development of pH-sensitive self-powered devices. Preliminary results with a non-buffered amplification kit pointed towards the feasibility of detecting the pH shift taking place upon NA amplification. However, further research to confirm this result is still needed.
2) The market landscape for a molecular rapid test has been widely explored. The main market entries for a test have been identified, as well as the most favorable countries to introduce a novel technology. Two market segment have been clearly identified to be the most suitable to have a successful market penetration: lyme disease and papilloma virus.
3) The evolution of the business initiatives aimed at producing the first generation of molecular tests has been closely followed. After the SARS-Covid pandemics, most of the companies have not achieved the deployment of their solutions to the market, which seems to experience a sever cooling down. However, there continues to be a need for a rapid diagnostic test that can detect the presence of pathogens with greater sensitivity and specificity than lateral flow-based tests.
4) One of the main issues in achieving a successful device is the high cost of the cassette. Thus, one of the main challenges is to integrate the cell lysis and molecular amplification steps in a smart way, with the minimum number of components. Therefore, one way to capitalize on the technical and commercial knowledge acquired throughout the proposal is to focus research on achieving this goal, while the development of a digital readout (the main objective of PRONTO) could be addressed at a later stage. A hybrid solution, where the result is interpreted by a lateral flow strip, would already represent a revolutionary breakthrough.
2) The market landscape for a molecular rapid test has been widely explored. The main market entries for a test have been identified, as well as the most favorable countries to introduce a novel technology. Two market segment have been clearly identified to be the most suitable to have a successful market penetration: lyme disease and papilloma virus.
3) The evolution of the business initiatives aimed at producing the first generation of molecular tests has been closely followed. After the SARS-Covid pandemics, most of the companies have not achieved the deployment of their solutions to the market, which seems to experience a sever cooling down. However, there continues to be a need for a rapid diagnostic test that can detect the presence of pathogens with greater sensitivity and specificity than lateral flow-based tests.
4) One of the main issues in achieving a successful device is the high cost of the cassette. Thus, one of the main challenges is to integrate the cell lysis and molecular amplification steps in a smart way, with the minimum number of components. Therefore, one way to capitalize on the technical and commercial knowledge acquired throughout the proposal is to focus research on achieving this goal, while the development of a digital readout (the main objective of PRONTO) could be addressed at a later stage. A hybrid solution, where the result is interpreted by a lateral flow strip, would already represent a revolutionary breakthrough.